IC50: 0.68 nM (Bcr-AblT315I), 0.15 nM (Bcr-AblQ252H), 0.27 nM (Bcr-AblE255K) , 0.29 nM (Bcr-Abl M351T), 0.35 nM (Bcr-Abl H396P), 0.71 nM (Bcr-AblG250E) , 0.35 nM (Bcr-AblY253F), Bcr-AblF317L[1]

In stably transformed Ba/F3 cells, Olverembatinib dimesylate shows antiproliferative activity, with growth driven by either native Bcr-Abl or Bcr-Abl mutants [1]. Leukemia cells positive for Bcr-Abl[1]. In K562 (1–20 nM; 4.0 hours) and Ba/F3 stable cell lines expressing native Bcr–Abl (0.1–100 nM; 4.0 hours) or Bcr–AblT315I (0.1–100 nM; 4.0 hours), olverembatinib dimesylate inhibits Bcr–Abl signaling[1].

In mice bearing allogeneic Ba/F3 cells expressing Bcr-AblWT, olverembatinib dimesylate reduces tumor growth[1]. The median lifespan of mice harboring allogeneic Ba/F3 cells expressing Bcr-AblT315I is markedly increased by olverembatinib dimesylate (1–20 mg/kg; ig; daily; for 10 days) [1]. Olverembatinib dimesylate, when administered orally to rats at a dose of 25 mg/kg, demonstrates a good oral bioavailability (rats 48.7%) and Cmax (rats 390.5 μg/L) [1]. Because of its high plasma clearance (1.7 L/h/kg) upon intravenous dosing (rat 5 mg/kg), olverembatinib dimesylate has a terminal elimination half-life (rat 5.6 hours) [1].

Active-Site-Dependent Competition Binding Assay. KINOMEscan Screening[1]
The binding activities of 10a with native Abl or Abl mutants were analyzed by KINOMEscan system conducted by Ambit Bioscience. Briefly, kinases were tagged with DNA. The ligands were biotinylated and immobilized to streptavidin-coated beads. The binding reactions were assembled by incubating DNA-tagged kinases, immobilized ligands, and test compounds in binding reactions (20% SeaBlock, 0.17 × PBS, 0.05% tween-20, 6 mM DTT) for 1.0 h at room temperature. The affinity beads were washed with washing buffer (1 × PBS, 0.05% Tween-20) first and then elution buffer (1 × PBS, 0.05% Tween 20, 0.5 μM nonbiotinylated affinity ligands). The kinase concentration in the eluate was determined by quantitative PCR of the DNA tagged to the kinase. The ability of the test compound to bind to the kinase was evaluated with percent control (%) as (test compound signal – positive control signal)/negative control signal – positive control signal) × 100%. Negative control is DMSO control (100% ctrl) and positive control is control compound (0% ctrl).

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FRET-Based Z′-Lyte Assay Detecting Peptide Substrate Phosphorylation[1]
The kinases were commercially purchased from Invitrogen. The catalog numbers of kinases ABL1, ABL1(E255K), ABL1 (G250E), ABL1(T315I), and ABL1(Y253F) are P3049, PV3864, PV3865, PV3866, and PV3863, respectively. All of them are full-length human recombinant protein expressed in insect cells and histidine-tagged. Inhibition activities of inhibitors against Abl1 and its mutants were performed in 384-well plates using the FRET-based Z′-Lyte assay system according to the manufacturer’s instructions. Briefly, the kinase substrate was diluted into 5 μL of 1X kinase reaction buffer; and the kinase was added. Compounds (10 nL) with indicated concentrations were then delivered to the reaction by using Echo liquid handler, and the mixture was incubated for 30 min at room temperature. Then 5 μL of 2X ATP solution was added to initiate the reaction, and the mixture was further incubated for 2 h at room temperature. The resulting reactions contained 10 μM (for wild-type Abl1, and mutants Y253F, Q252H, M351T, and H396P) or 5 μM (for mutants E255K, G250E, T315I) of ATP, 2 μM of Tyr2 Peptide substrate in 50 mM HEPES (PH 7.5), 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA, 0.0247 μg/mL Abl1, and inhibitors as appropriate. Then, 5 μL of development reagent was added, and the mixture was incubated for 2 h at room temperature before 5 μL of stop solution was added. Fluorescence signal ratio of 445 nm (Coumarin)/520 nm (fluorescin) was examined on an EnVision Multilabel Reader. The data were analyzed using Graphpad Prism5. The data were the mean value of three experiments.

Western Blot Analysis[1]
Cell Types: K562 cells
Tested Concentrations: 1 nM, 2 nM, 5 nM, 10 nM, 20nM
Incubation Duration: 4.0 hrs (hours)
Experimental Results: Inhibited Bcr-Abl signaling in K562 cell lines.

Animal/Disease Models: SCID nude mice, bearing allografted Ba/F3 cells expressing Bcr-AblT315I[1]
Doses: 1 mg/kg, 2 mg/kg, 5.0 mg/kg, 10 mg/kg, 20 mg/kg
Route of Administration: po (oral gavage) , daily, for 10 days
Experimental Results: Efficiently prolonged animal survival in an allograft leukemia tumor model.

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Animal/Disease Models: Rats[1]
Doses: 5 mg/kg for iv; 25 mg/kg for oral (pharmacokinetic/PK Analysis)
Route of Administration: intravenous (iv) injection and oral administration
Experimental Results: Oral bioavailability (48.7%), Cmax (390.5 μg/L), T1/2 (5.6 h).

[1]. Ren X, Pan X, Zhang Z, Identification of GZD824 as an orally bioavailable inhibitor that targets phosphorylated and nonphosphorylated breakpoint cluster region-Abelson (Bcr-Abl) kinase and overcomes clinically acquired mutation-induced resistance against imatinib. J Med Chem. 2013 Feb 14;56(3):879-94.

Bcr-Abl(T315I) mutation-induced imatinib resistance remains a major challenge for clinical management of chronic myelogenous leukemia (CML). Herein, we report GZD824 (10a) as a novel orally bioavailable inhibitor against a broad spectrum of Bcr-Abl mutants including T315I. It tightly bound to Bcr-Abl(WT) and Bcr-Abl(T315I) with K(d) values of 0.32 and 0.71 nM, respectively, and strongly inhibited the kinase functions with nanomolar IC(50) values. The compound potently suppressed proliferation of Bcr-Abl-positive K562 and Ku812 human CML cells with IC(50) values of 0.2 and 0.13 nM, respectively. It also displayed good oral bioavailability (48.7%), a reasonable half-life (10.6 h), and promising in vivo antitumor efficacy. It induced tumor regression in mouse xenograft tumor models driven by Bcr-Abl(WT) or the mutants and significantly improved the survival of mice bearing an allograft leukemia model with Ba/F3 cells harboring Bcr-Abl(T315I). GZD824 represents a promising lead candidate for development of Bcr-Abl inhibitors to overcome acquired imatinib resistance.[1]

C29H27F3N6O.2CH4O3S

724.77

628.208

C, 51.37; H, 4.87; F, 7.86; N, 11.60; O, 15.45; S, 8.85

1421783-64-3

Olverembatinib;1257628-77-5

71519689

Typically exists as light yellow to yellow solids at room temperature

5.23

139.9

CC1C=CC(C(NC2C=CC(CN3CCN(C)CC3)=C(C(F)(F)F)C=2)=O)=CC=1C#CC1C=NC2NN=CC=2C=1.CS(O)(=O)=O.CS(O)(=O)=O

LEVIGHXVOVROGW-UHFFFAOYSA-N

InChI=1S/C29H27F3N6O.2CH4O3S/c1-19-3-5-22(14-21(19)6-4-20-13-24-17-34-36-27(24)33-16-20)28(39)35-25-8-7-23(26(15-25)29(30,31)32)18-38-11-9-37(2)10-12-38;2*1-5(2,3)4/h3,5,7-8,13-17H,9-12,18H2,1-2H3,(H,35,39)(H,33,34,36);2*1H3,(H,2,3,4)

CC1=C(C=C(C=C1)C(=O)NC2=CC(=C(C=C2)CN3CCN(CC3)C)C(F)(F)F)C#CC4=CC5=C(NN=C5)N=C4.CS(=O)(=O)O.CS(=O)(=O)O

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 2.08 mg/mL (2.87 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (2.87 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (2.87 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: Saline: 20mg/mL

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 (Please use freshly prepared in vivo formulations for optimal results.)